Objective: To observe the mutagenicity of retrovirus and adenovirus as transgenic vectors to evaluate the safety of transgenic tumor cells as tumor vaccines. Methods: Cells were cultured together with the virus. Then ...Objective: To observe the mutagenicity of retrovirus and adenovirus as transgenic vectors to evaluate the safety of transgenic tumor cells as tumor vaccines. Methods: Cells were cultured together with the virus. Then DNA and supernatant were tested for mutagenicity by means of genetic toxicological laboratory technique. Results: The results indicated that DNA and supernatant of transgenic cells had no mutagenicity through both in vivo and in vitro tests. Conclusion: The modified virus had no mutagenicity as a transgenic vector.展开更多
Targeted genome editing technology has been widely used in biomedical studies. The CRISPR- associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for study...Targeted genome editing technology has been widely used in biomedical studies. The CRISPR- associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio- medicine.展开更多
Gaucher disease (GD) is an autosomal recessive disease which if undiagnosed or diagnosed late results in devastating complications. Because of the heterozygous nature of GD, there is a wide spectrum of clinical presen...Gaucher disease (GD) is an autosomal recessive disease which if undiagnosed or diagnosed late results in devastating complications. Because of the heterozygous nature of GD, there is a wide spectrum of clinical presentation. Clinicians should be aware of this rare but potentially treatable disease in patients who present with unexplained organomegaly, anemia, massive splenomegaly, ascites and even cirrhosis of unknown origin. The treatment options for adult type GD include enzyme replacement treatment (ERT) and substrate reduction treatment (SRT) depending on the status of the patient. Future treatment options are gene therapy and "smart molecules" which provide specifi c cure and additional treatment options. In this review, we present the key issues about GD and new developments that gastroenterologists should be aware of.展开更多
Xerostomia is the main complication inflicting head and neck cancer patients treated with radiotherapy.Conventional treatment is not very effective in alleviating this morbidity.Biomedical strategies such as gene tran...Xerostomia is the main complication inflicting head and neck cancer patients treated with radiotherapy.Conventional treatment is not very effective in alleviating this morbidity.Biomedical strategies such as gene transfer and tissue engineering have made substantial progress that will potentially lead to successful new treatment options for this condition.This report reviews the process of radiation damage to the salivary glands and the advances in functional salivary gland enhancement with these two brand-new technologies.展开更多
Objective:Cytopathic effects and local immune response were analyzed histologically in prostatic cancer(PCa)with in situ herpes simplex virus-thymidine kinase(HSV-tk)/ganciclovir(GCV)gene therapy(GT).Methods:Four high...Objective:Cytopathic effects and local immune response were analyzed histologically in prostatic cancer(PCa)with in situ herpes simplex virus-thymidine kinase(HSV-tk)/ganciclovir(GCV)gene therapy(GT).Methods:Four high-risk PCa patients who received HSV-tk/GCV GT were investigated.After two cycles of intraprostatic injection of HSV-tk and administration of GCV,radical prostatectomy was performed.Formalin-fixed,paraffin-embedded sections were evaluated using immunohistochemistry.PCa with hormone therapy(HT,n=3)or without neoadjuvant therapy(NT,n=4)that were equivalent in terms of risk were also examined as reference.Immunoreactively-positive cells were counted in at least three areas in cancer tissue.Labeling indices(LI)were calculated as percentage values.Results:ssDNA LI in GT increased,indicating apoptosis,as well as tumor-infiltrating lymphocytes and CD68-positive macrophages,compared with their biopsies.GT cases showed significantly higher numbers of single-stranded DNA(ssDNA)LI,CD4/CD8-positive T cells and CD68-positive macrophages including M1/M2 macrophages than HT or NT cases.However,there was no significant difference in CD20-positive B cells among the types of case.There were strong correlations between CD8+T cells and CD68+macrophages(ρ=0.656,p<0.0001)as well as CD4+T cells and CD20+B cells(ρ=0.644,p<0.0001)in PCa with GT.Conclusions:Enhanced cytopathic effect and local immune response might be indicated in PCa patients with HSV-tk/GCV gene therapy.展开更多
Objective: to transduce the tumor associated antigen gene MAGE-1 and/or IL-12 gene into dendritic cells (DC) and to observe the in vitro cytotoxic effect induced by the genetically modified DC against the human hepato...Objective: to transduce the tumor associated antigen gene MAGE-1 and/or IL-12 gene into dendritic cells (DC) and to observe the in vitro cytotoxic effect induced by the genetically modified DC against the human hepatocellular carcinoma (HCC) cell line SMMC7721. Methods: the MAGE-1 gene was inserted into the retrovirus vector LXSN to construct the recombinant retrovirus LMSN. The monocyte-derived DCs were transfected at appropriate differentiation stage by LMSN and/or a recombinant adenovirus AdmiL-12, which containing murine IL-12 gene. The control groups included retrovirus LXSN transfected, adenovirus AdBGFP transfected and non-transfected DCs. The MAGE-1 gene expression was identified by western blot and the mIL-12 p70 secretion was detected by ELISA assay. The in vitro cytotoxicities against SMMC7721 induced by genetically modified and control groups of DC were tested by MTT assay. Results: The MAGE-1 expression was detected by a monoclonal antibody in DCs tranfected with LMSN but not in control groups. At 16 h, 24 h and 48 h after transfection with AdmIL-12, the concentration of the mIL-12 p70 in the culture medium was 580pg/106 cells, 960pg/106 cells and 1100pg/106 cells respectively. The mIL-12 p70 secretions were not detected in other groups. The lytic activity (as judged by % lysis) induced by each groups of DC was 94.2±5.2% (LMSN and AdmIL-12 cotransfected group), 78.9±3.6% (LMSN transfected groups), 52.6±9.7% (AdmIL-12 transfected group), 34.7±4.3% (LXSN transfected group), 36.3±3.8% (AdBGFP transfected group) and 3.9±2.0% (non-transfected group) respectively. Except for LXSN transfected and AdBGFP transfected group, the difference of the lytic activities between other groups were statistically significant (P<0.05). Conclusion: The MAGE-1 gene modified DCs can induce relatively specific cytotoxicty against SMMC7721 in vitro and thus suggested that those genetically engineered DCs have the potential to serve as novel vaccine for HCC. Transduction of exogenous IL-12 gene into DCs may further enhance DCs’ activity and the effectiveness of the above DC vaccine.展开更多
Objective To investigate the effects of combination adenovirusmediated HSVtk/GCV system and antisense IGF1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV kill...Objective To investigate the effects of combination adenovirusmediated HSVtk/GCV system and antisense IGF1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSVtk and antisense IGF1 was observed in vitro.Rat glioma was treated with HSVtk/GCV and antisense IGF1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+ 4,CD+ 8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF1 gene may enhance the tumorkilling effects of HSVtk/GCV.展开更多
Recombinant adenovirus(rAdV)is a commonly used vector system for gene transfer.Efficient initial packaging and subsequent production of rAdV remains time-consuming and labor-intensive,possibly attributable to rAdv inf...Recombinant adenovirus(rAdV)is a commonly used vector system for gene transfer.Efficient initial packaging and subsequent production of rAdV remains time-consuming and labor-intensive,possibly attributable to rAdv infection-associated oxidative stress and reactive oxygen species(ROS)production.Here,we show that exogenous GAPDH expression mitigates adenovirus-induced ROS-associated apoptosis in HEK293 cells,and expedites adenovirus production.By stably overexpressing GAPDH in HEK293(293G)and 293pTP(293GP)cells,respectively,we demonstrated that rAdV-induced RoS production and cell apoptosis were significantly suppressed in 293G and 293GP cells.Transfection of 293G cells with adenoviral plasmid pAd-G2Luc yielded much higher titers of Ad-G2Luc at day 7 than that in HEK293 cells.Similarly,Ad-G2Luc was amplified more efficiently in 293G than in HEK293 cells.We further showed that transfection of 293GP cells with pAd-G2Luc produced much higher titers of Ad-G2Luc at day 5 than that of 293pTP cells.293GP cells amplified the Ad-G2Luc much more efficiently than 293pTP cells,indicating that exogenous GAPDH can further augment pTP-enhanced adenovirus production.These results demonstrate that exogenous GAPDH can effectively suppress adenovirus-induced ROS and thus accelerate adenovirus production.Therefore,the engineered 293GP cells represent a superfast rAdV production system for adenovirus-based gene transfer and gene therapy.展开更多
基金Foundation item: This work was supported by a grant from the Natural Science Foundation of Heilongjiang Province (D0122).
文摘Objective: To observe the mutagenicity of retrovirus and adenovirus as transgenic vectors to evaluate the safety of transgenic tumor cells as tumor vaccines. Methods: Cells were cultured together with the virus. Then DNA and supernatant were tested for mutagenicity by means of genetic toxicological laboratory technique. Results: The results indicated that DNA and supernatant of transgenic cells had no mutagenicity through both in vivo and in vitro tests. Conclusion: The modified virus had no mutagenicity as a transgenic vector.
基金partially supported by the National Natural Science Foundation of China(81202110,81120108019,U1132605 and 81325016)
文摘Targeted genome editing technology has been widely used in biomedical studies. The CRISPR- associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio- medicine.
文摘Gaucher disease (GD) is an autosomal recessive disease which if undiagnosed or diagnosed late results in devastating complications. Because of the heterozygous nature of GD, there is a wide spectrum of clinical presentation. Clinicians should be aware of this rare but potentially treatable disease in patients who present with unexplained organomegaly, anemia, massive splenomegaly, ascites and even cirrhosis of unknown origin. The treatment options for adult type GD include enzyme replacement treatment (ERT) and substrate reduction treatment (SRT) depending on the status of the patient. Future treatment options are gene therapy and "smart molecules" which provide specifi c cure and additional treatment options. In this review, we present the key issues about GD and new developments that gastroenterologists should be aware of.
文摘Xerostomia is the main complication inflicting head and neck cancer patients treated with radiotherapy.Conventional treatment is not very effective in alleviating this morbidity.Biomedical strategies such as gene transfer and tissue engineering have made substantial progress that will potentially lead to successful new treatment options for this condition.This report reviews the process of radiation damage to the salivary glands and the advances in functional salivary gland enhancement with these two brand-new technologies.
基金supported by Grants-in-Aid for Scientific Research(JSPS KAKENHI)grant(number 21592060).
文摘Objective:Cytopathic effects and local immune response were analyzed histologically in prostatic cancer(PCa)with in situ herpes simplex virus-thymidine kinase(HSV-tk)/ganciclovir(GCV)gene therapy(GT).Methods:Four high-risk PCa patients who received HSV-tk/GCV GT were investigated.After two cycles of intraprostatic injection of HSV-tk and administration of GCV,radical prostatectomy was performed.Formalin-fixed,paraffin-embedded sections were evaluated using immunohistochemistry.PCa with hormone therapy(HT,n=3)or without neoadjuvant therapy(NT,n=4)that were equivalent in terms of risk were also examined as reference.Immunoreactively-positive cells were counted in at least three areas in cancer tissue.Labeling indices(LI)were calculated as percentage values.Results:ssDNA LI in GT increased,indicating apoptosis,as well as tumor-infiltrating lymphocytes and CD68-positive macrophages,compared with their biopsies.GT cases showed significantly higher numbers of single-stranded DNA(ssDNA)LI,CD4/CD8-positive T cells and CD68-positive macrophages including M1/M2 macrophages than HT or NT cases.However,there was no significant difference in CD20-positive B cells among the types of case.There were strong correlations between CD8+T cells and CD68+macrophages(ρ=0.656,p<0.0001)as well as CD4+T cells and CD20+B cells(ρ=0.644,p<0.0001)in PCa with GT.Conclusions:Enhanced cytopathic effect and local immune response might be indicated in PCa patients with HSV-tk/GCV gene therapy.
基金This work was supported by NationalNatural Science Foundation of China (No. 39770826) andpartly by CMB (No. 93583).
文摘Objective: to transduce the tumor associated antigen gene MAGE-1 and/or IL-12 gene into dendritic cells (DC) and to observe the in vitro cytotoxic effect induced by the genetically modified DC against the human hepatocellular carcinoma (HCC) cell line SMMC7721. Methods: the MAGE-1 gene was inserted into the retrovirus vector LXSN to construct the recombinant retrovirus LMSN. The monocyte-derived DCs were transfected at appropriate differentiation stage by LMSN and/or a recombinant adenovirus AdmiL-12, which containing murine IL-12 gene. The control groups included retrovirus LXSN transfected, adenovirus AdBGFP transfected and non-transfected DCs. The MAGE-1 gene expression was identified by western blot and the mIL-12 p70 secretion was detected by ELISA assay. The in vitro cytotoxicities against SMMC7721 induced by genetically modified and control groups of DC were tested by MTT assay. Results: The MAGE-1 expression was detected by a monoclonal antibody in DCs tranfected with LMSN but not in control groups. At 16 h, 24 h and 48 h after transfection with AdmIL-12, the concentration of the mIL-12 p70 in the culture medium was 580pg/106 cells, 960pg/106 cells and 1100pg/106 cells respectively. The mIL-12 p70 secretions were not detected in other groups. The lytic activity (as judged by % lysis) induced by each groups of DC was 94.2±5.2% (LMSN and AdmIL-12 cotransfected group), 78.9±3.6% (LMSN transfected groups), 52.6±9.7% (AdmIL-12 transfected group), 34.7±4.3% (LXSN transfected group), 36.3±3.8% (AdBGFP transfected group) and 3.9±2.0% (non-transfected group) respectively. Except for LXSN transfected and AdBGFP transfected group, the difference of the lytic activities between other groups were statistically significant (P<0.05). Conclusion: The MAGE-1 gene modified DCs can induce relatively specific cytotoxicty against SMMC7721 in vitro and thus suggested that those genetically engineered DCs have the potential to serve as novel vaccine for HCC. Transduction of exogenous IL-12 gene into DCs may further enhance DCs’ activity and the effectiveness of the above DC vaccine.
文摘Objective To investigate the effects of combination adenovirusmediated HSVtk/GCV system and antisense IGF1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSVtk and antisense IGF1 was observed in vitro.Rat glioma was treated with HSVtk/GCV and antisense IGF1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+ 4,CD+ 8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF1 gene may enhance the tumorkilling effects of HSVtk/GCV.
基金supported in part by research grants from the Natural Science Foundation of China (No.82000744 to ZT,and 82102696 to J.F.)the Chongqing Bayu Young Scholar Award (China) (to J.F.)+5 种基金the 2019 Funding for Postdoctoral Research (Chongqing Human Resources and Social Security Bureau of China) (No.298 to J.F.)the National Institutes of Health (No.CA226303 to T.C.H.,DE030480 to R.R.R.)supported by the Medical Scientist Training Program of the National Institutes of Health (USA) (No.T32 GM007281)supported in part by The University of Chicago Cancer Center Support Grant (No.P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through grant number 2UL1TR002389-06 that funds the Institute for Translational Medicine (ITM)supported by the Mabel Green Myers Research Endowment Fund and The University of Chicago Orthopaedics Alumni Fund.
文摘Recombinant adenovirus(rAdV)is a commonly used vector system for gene transfer.Efficient initial packaging and subsequent production of rAdV remains time-consuming and labor-intensive,possibly attributable to rAdv infection-associated oxidative stress and reactive oxygen species(ROS)production.Here,we show that exogenous GAPDH expression mitigates adenovirus-induced ROS-associated apoptosis in HEK293 cells,and expedites adenovirus production.By stably overexpressing GAPDH in HEK293(293G)and 293pTP(293GP)cells,respectively,we demonstrated that rAdV-induced RoS production and cell apoptosis were significantly suppressed in 293G and 293GP cells.Transfection of 293G cells with adenoviral plasmid pAd-G2Luc yielded much higher titers of Ad-G2Luc at day 7 than that in HEK293 cells.Similarly,Ad-G2Luc was amplified more efficiently in 293G than in HEK293 cells.We further showed that transfection of 293GP cells with pAd-G2Luc produced much higher titers of Ad-G2Luc at day 5 than that of 293pTP cells.293GP cells amplified the Ad-G2Luc much more efficiently than 293pTP cells,indicating that exogenous GAPDH can further augment pTP-enhanced adenovirus production.These results demonstrate that exogenous GAPDH can effectively suppress adenovirus-induced ROS and thus accelerate adenovirus production.Therefore,the engineered 293GP cells represent a superfast rAdV production system for adenovirus-based gene transfer and gene therapy.
